The relevance of migraine in the clinical spectrum of mitochondrial disorders

Recent scientific evidence suggests a link between migraine and brain energy metabolism. In fact, migraine is frequently observed in mitochondrial disorders. We studied 46 patients affected by mitochondrial disorders, through a headache-focused semi-structured interview, to evaluate the prevalence of migraine among patients affected by mitochondrial disorders, the possible correlations between migraine and neuromuscular genotype or phenotype, comorbidities, lactate acid levels and brain magnetic resonance spectroscopy. We explored migraine-related disability, analgesic and prophylactic treatments. Diagnoses were achieved according to International Classification of Headache Disorders, 3rd edition. Lifetime prevalence of migraine was 61% (28/46), with high values in both sexes (68% in females, 52% in males) and higher than the values found in both the general population and previous literature. A maternal inheritance pattern was reported in 57% of cases. MIDAS and HIT6 scores revealed a mild migraine-related disability. The high prevalence of migraine across different neuromuscular phenotypes and genotypes suggests that migraine itself may be a common clinical manifestation of brain energy dysfunction. Our results provide new relevant indications in favour of migraine as the result of brain energy unbalance.

. In this context, "Stroke/TIA" refers to an actual cerebrovascular events, i.e.: stroke in case of clear ischemic bran lesions, and TIA in case of transient acute neurological deficit in patients with multiple cerebrovascular risk factors. The interview was conducted face-to-face in 57% of cases (26/46), and by telephone in 43% (20/46). A total of 21/46 patients (45.7%) reported at least one first-grade or second-grade relative who had suffered from recurrent headaches. In this group, a maternal inheritance pattern was reported in 12/21 cases (57%); paternal inheritance in two cases, while siblings but not parents were affected in the remaining seven cases (33%).
Considering the wide age range (17-83 years) and the high mean age of our patients cohort, data about the lifetime prevalence of migraine are more relevant: 18/46 (39.1%) of patients achieved the diagnosis of MO (in one case together with MA diagnosis), one reported CM, 2/46 (4.3%) had MA, in one case with hemiplegic features.  Figure 1. Prevalence of primary headaches among patients with mitochondrial disorders, according to ICHD3 criteria. One-year prevalence (lower bar); lifetime prevalence of migrainous headache (including migraine without aura, migraine with aura, chronic migraine and probable migraine without aura) by sex (first and second bars) and in the whole study population (third bar).
A group of 7/46 patients (15.2%) reported pMO. Among the pMO patients, diagnostic criterion D ("during headache at least one of the following: 1. Nausea and/or vomiting; 2. Photophobia and phonophobia" 16 ) was lacking in four cases. Criteria B ("headache attacks lasting 4-72 h, when untreated or unsuccessfully treated" 16 ) and C ("headache has al least two of the following four characteristics: 1. Unilateral location, 2. Pulsating quality, 3. Moderate or severe pain intensity, 4. Aggravation by or causing avoidance of routine physical activity" 16 ) were not satisfied in two and one cases, respectively. Overall, 60.9% (28/46) of patients with a mitochondrial disorder suffered from a migrainous headache in their lifetime. Migraine prevalence was 68.0% in females (17/25), and 52.4% (11/21) in male patients ( Fig. 1). Only 10/46 patients (21.7% of cases) did not report any sort of recurrent headache in their life, despite a higher mean age (66.0 ± 13.9 years; range 42-83 years).
Clinical features of headache in mitochondrial patients. The median age at onset of headaches was 25.5 years, with 95% confidence interval from 20 to 40 years ( Fig. 2A); this model includes "censored" patients who never developed headache. Migraine showed a median clinical onset at about 20 years, where TTH patients had an earlier median onset, at 19 years (Fig. 2B,C); these headache-type-specific models excluded "censored" patients, hence the earlier estimated median values. The difference between the median age at onset among the different headache subtypes was not statistically significant. Nine patients reported the past resolution of a highly-disabling migrainous headache towards a less impacting ETTH (5 cases) at the mean age of 44 years or a complete resolution of their MO (2 cases) or pMO (1 case), at a mean age of 51 years. One patient reverted from CM to MO, while another one stopped suffering from MO, while continuing with his MA attacks. Aura prevalence reported in only 3 patients (10.7% of the migraineurs): 2 were typical auras, while 1 patient carrying MICU1 mutations experienced hemiplegic migraine.
Our 28 migrainous patients had experienced, at least one, 64% (42/66) of the triggers listed in our semistructured 66-item-questionnaire, including the most frequent triggers of migrainous headache, according to specific scientific literature [17][18][19] . Each patient reported up to 15 different triggers of migraine. As expected, among the most reported factors there were stress (39%), work-related stress (36%) or family-related stress (29%). More frequently, 54% of migrainous patients could had attacks triggered by sleeping deprivation, 11% with sleeping more than usual or 18% in case of sleeping in the afternoon. 53% of female migraineurs pointed to female hormonal fluctuations (including premenstrual period, menstrual phase, ovulatory period or oral contraceptives) (see Table 2 for the complete list).
The quality of pain among the 28 migraineurs was throbbing in 64% (18/28) of cases, with a mean headache intensity of 8.25/10 (Number Rating Scale). It was reported as unilateral in 46% (13/28) of cases, bilateral in 54% (15/28), with predominant origin in the frontal area (36%-10/28) and irradiation to the homolateral half   , with a median value of 2.5 attacks per month. 12 patients (43%) reported more than three migraine attacks per month that were often moderately or severely disabling (75% of these sub-sample). Two other patients reported a mean of three highly disabling www.nature.com/scientificreports/ migraine attacks per month, whose duration may reach the 48 h. 50% of our active migraineurs did benefit from a prophylactic therapy, but none was regularly assuming it. Considering the whole sample, only 18% (5/28) of our migrainous patients had tried a preventive therapy at least once in their life, including flunarizine (3 cases), followed by topiramate, pizotifen, propranolol, acupuncture and nutraceutical (1 case each).  Fig. 3a). The absence of any primary headaches results associated with CPEO phenotype with statistical significance (p = 0.036). The age of onset of headache was not significantly different across various phenotypic groups (Fig. 2d). A total of 32/46 patients (70%) had a defined genotype. 11 patients harboured nDNA mutations and represented the largest group: POLG mutations were identified in 7, and OPA1, DGUOK, MGME1 and MICU1 gene mutation in one patient each. Six patients had multiple mtDNA deletions but no nuclear gene mutation identified yet. Thirteen patients showed a single mtDNA deletion, while eight had a mtDNA point mutation: A3243G in 6, a tRNA Leu (mt.C3250T) and tRNA Met (mt.A4415G) in in one patient each. 60.5% (23/38) of patients with a defined disease-causing mutation or with multiple mtDNA deletions suffered from a migrainous headache.      Fig. 3b). The distribution of primary headaches with the different genotypes did not differ significantly among subgroups. Age at headache onset and lack of primary headaches was not statistically different according to genotypes (Fig. 2e). Among 19 patients with active migraine, mean HIT6 score was 55.1 ± 6.8 pts (range 46-65). Eleven patients reported mild (HIT6 score between 50 and 55 points) or no disability (HIT6 score < 50 points) related to their migraine; eight (42%) experienced a moderate (HIT6 score from 56 to 59 points) or severe (HIT6 score > 59 points) migraine-related disability. CPEO plus patients showed a tendency towards a higher degree of disability and KSS patients reported a mild degree of disability, but no statistically significant differences in HIT6 scores could be found across different phenotypes or genotypes. POLG patients showed a tendency towards a higher migraine-related disability ( Supplementary Information 2).

Mitochondrial phenotype and genotype and migraine. Clinical phenotype (
The 19 patients with active migraine reported a mean MIDAS score of 11.2 ± 19.6 points (range 0-80 points) with a median of 1 point. A mild (MIDAS score from 6 to 10 points) or neglectable migraine-related disability (MIDAS score < 6 points) has been reported by 74% (14/19) of the patients, with only two cases of moderate disability (MIDAS score from 11 to 20 points) and three patients with severe disability related to migraine (MIDAS score > 20 points). The last five patients had a moderate-severe disability score at HIT6, as well.
Treatment strategies. the great majority of patients (79%-22/28) took non-steroidal anti-inflammatory drugs (NSAIDs), with paracetamol and ibuprofen as the leading drugs (29% of cases, each), combination-analgesic or metamizole in three cases, while three patients (11%) did not assume any acute treatment for their migraine attacks. None of our 28 mitochondrial patients treated their migraine with triptans or ergot alkaloids, even if two patients (7% of migraineurs) reported previous Medication Overuse Headache (one paracetamol-overuse headache and a combination-analgesic overuse headache).

Discussion
Our 46 patients cross-sectional study represents the fourth largest study in scientific literature, regarding primary headaches in adult patients with mitochondrial disorders. We found a one-year prevalence of migrainous headache of 41%, significantly greater than the prevalence reported in general population in the European countries 20,21 , and higher than the 25.9% prevalence reported in a recent large Italian population study 22 . A total of 61% of mitochondrial disease patients suffered or had suffered of a migrainous headache. Since our sample has a high mean age and migraine prevalence is known to decrease with increasing age 20 , the prevalence in our patients' cohort is more than threefold higher than expected in the general population 20 in both sexes, reaching 68% in female. Our estimates are higher than those reported in a recent large Dutch study (55%, mainly focused on mt.3243A > G patients 23 ) and in a previous study with more heterogenous subtypes of mitochondrial patients (35.5% 24 ). Given the well-known high prevalence of migraine among mt.3243A > G patients 25,26 , the limited number of MELAS patients in our cohort minimizes selection bias. CPEO and CPEO plus are the main phenotypes in our study population, with a subgroup prevalence of migraine of 53%, which is notably higher than the 21-40% prevalence previously reported among CPEO patients [27][28][29] . We found higher migraine prevalence also in POLG patients (57 vs. 23%) 30 , whereas the prevalence was similar to that of literature in MELAS patients (67%) 26 . Age at onset, evolution over time, prodromal symptoms, trigger, features and duration of headache are in line with those of the general population with migraine, confirming that the clinical entity is likely the same. However, a few differences from the general population emerged. First, phonophobia shows 54% prevalence and osmophobia involves only 18% of migraineurs, which are frequencies lower than those literature 17,31 . This could be partially due to the frequent hearing loss (reported up to 43% of our cohort) or to the decrease in olfactory perception induced by mitochondrial pathology (nearly 10% of our sample). In fact, in our 7 pMO patients the diagnostic criterion D ("During headache at least one of the following: (1) nausea and/or vomiting; (2) photophobia and phonophobia" 16 ) was absent more often than other definitory features. Furthermore, the 11% prevalence of prodromal symptoms is significantly lower than the 72% reported in literature 10 , with a probable bias due to memory recall. These data suggest that we did not document a mere migraine-like headache: migraine, as coded in ICHD3, appears indeed a core clinical manifestation of the clinical spectrum of mitochondrial disorders.
Considering the familiar occurrence of migraine in general population, a maternal inheritance pattern is by far the most frequent 32,33 , providing another hint in favour of a possible role of mitochondria in migraine pathogenesis.
Interestingly, isolated stress (50%), lack of sleep (54%), oestrogen fluctuations (53%), fatigue (24%), vigorous physical exercise (18%), fasting or skipping meals (15%) are some of the consistently reported migraine triggers which can be connected to failure of energy metabolism, mitochondrial dysfunction and oxidative stress. Accordingly, and in contrast with previous literature data 24 , we observed a statistically significant association between migraine and increased blood lactate levels, likewise our 3 MO patients all had increased lactate levels detected by 1 H-MRS.
We failed to find any significant difference in age at onset and migraine prevalence, nor was migraine prevalence significantly different across various mitochondrial genotypes, with a prevalence ranging from 53% (nDNA mutation) to 75% (mtDNA point mutation). This lack of correlation between migraine and neuromuscular phenotype or genotype is in line with previous literature 24 and suggests that migraine is likely associated with dysfunction of brain energy metabolism, rather than with a specific mutation or clinical pattern. www.nature.com/scientificreports/ Migraine showed a statistically significant association with female sex and sleep disturbances (and a tendency towards a correlation with increasing BMI), in accordance with previous studies on migraine [34][35][36][37] , strengthening the theorized central role of energy unbalance as the fulcrum of migraine pathogenesis.
Concerning the the results of HIT6 and MIDAS scales, the mean values reported by the only previous work on migraine in mitochondrial disorders 23 point to a mild level of disability. In our study, 42% of our activemigraine individuals reported an at least moderate migraine-related impairment on HIT6 scale, compared to the 62% prevalence found by Tiehuis and collaborators in a younger Dutch population 23 . 26% of our patients had a disabling migraine according to MIDAS, versus 34% in the scientific literature 23 . No association between HIT6/MIDAS scores and mitochondrial phenotypes or genotypes has been found. However, "CPEO plus" and POLG patients showed a tendency towards a higher degree of disability.
Data on migraine treatment were surprising. None of our patients was being administered a preventive therapy and only 18% of patients with a lifetime diagnosis of migrainous headache had tried prophylaxis before our examination, nor acute treatment was previously considered: no one had tried triptans in their life, whereas common analgesics were commonly used in a few cases. Treatment of migraine was far less than optimal in patients with mitochondrial disorders, often without exploiting the most appropriated available pharmaceutical tools. This under-treatment of migraine is in line with the few available data in scientific literature 23 : it could be partially due to the (wrong) underevaluation, both by patients and physicians, of the role played by headache in the syndromic output of the patients, when compared to the systemic and sometimes life-threatening dysfunctions associated with myopathy or mitochondrial pathology.
Our study sheds light on the role of migraine in mitochondrial diseases, with some limitations. First, the crosssectional design of our study may introduce a recall bias. Second, the still limited size of our cohort of patients makes it difficult to reach statistically sound associations between migraine and neuromuscular phenotypes or genotypes. Third, we lack a matched control group for a direct comparison. Fourth, we did not perform any measure of cognitive and/or memory performance in order to quantify the consistency and completeness of reported data, and finally, psychiatric comorbidities have not been considered in our study.
In this observational study, our preliminary clinical data converge to define migraine and its subtypes as a typical clinical manifestation of mitochondrial disorders. The lifetime prevalence of migraine was 61%, which is markedly higher than the values found in both the general population and the previous literature concerning mitochondrial disorders. In agreement with a previous article 24 , the prevalence of migraine was high across different neuromuscular phenotypes and genotypes suggesting that migraine itself may be a common clinical manifestation of brain energy dysfunction. Our results provide new relevant indications in favour of migraine as the result of brain energy unbalance.

Materials and methods
Ethical approval. This study was approved by the "Ethical Committee for Clinical Studies of the Padova district " and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Patients.
We performed an observational study on a series of patients affected by mitochondrial disorders followed at the Neuromuscular Center of the Neurological Clinic of the University of Padova. We included patients with a genetically confirmed diagnosis of mitochondrial disorder and patients in whom the combination of clinical (muscle w/o multisystem involvement), biochemical (OXPHOS dysfunction), neuroimaging (MR abnormalities) and histopathological (ragged red fibers, COX negative/SDH positive fibers, subsarcolemmal rims at SDH) data could strongly suggest the diagnosis of mitochondrial encephalomyopathy, even in absence of a known pathogenic mutation. Patients were clinically classified in "CPEO" (progressive bilateral ptosis and ophthalmoparesis), "KSS" (Kearns-Sayre syndrome, early onset CPEO plus cardiac conduction block and pigmentary retinopathy w/o multisystem involvement), "CPEO plus" (CPEO associated to multiple features of neuromuscular and multisystem involvement), autosomal dominant optic atrophy ("ADOA"), mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes ("MELAS") and other mitochondrial myopathy ("MM"). Patients were genotypically classified in individuals carrying nuclear DNA (nDNA) mutations, mitochondrial DNA (mtDNA) mutations and accumulation of multiple mtDNA deletions. All patients underwent neurological examination. Brain MRS and blood lactate levels were collected in a subset of patients.

Headache evaluation.
A semi-structured headache-focused interview was performed by a neurologist with specific training in the headache field; whenever possible it was carried out face-to-face, otherwise a telephone interview was performed. We asked for relatives suffering from headache, the time of onset of headache in the personal medical history and its evolution over time, the prodromal and associated symptoms, possible triggers (through a detailed 66-items semi-structured retrospective questionnaire Supplementary Information 4) and therapy. MIDAS and HIT6 scales were administered to each patient to measure migraine-related disability. Diagnoses of primary headache were codified according to International Criteria of Headache Disorders 3rd edition (ICHD3) criteria 16 . Possible correlations between migraine and neuromuscular phenotype or genotype were investigated. All patients had given written informed consent to the scientific use of their clinical data, which have been treated in a completely anonymous way. We excluded patients aged < 16 years or with severe cognitive decline and no reliable caregiver that made impossible the administration of the semi-structured interview, patients with intracranial space-occupying lesion or with a history of recent (less < 1 year) stroke.
Statistical analyses. Results were displayed as mean and standard deviation (SD) or median and range when appropriate. Age at headache onset was reported using the Kaplan-Meier method, and compared between